A Novel Sporothrix Brasiliensis Genomic Variant in Midwestern Brazil

medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

1 A novel Sporothrix brasiliensis genomic variant in Midwestern Brazil: 2 evidence for an older and wider sporotrichosis outbreak 3 4 João Eudes Filhoa, Isabele Barbieri Santosb, Carmélia Matos Santiago Reisc, José Salvatori 5 Patanéd, Verenice Paredese, João Paulo Romualdoe, Sabrina Santos Costa Poggiannif, Talita de 6 Cássia Borges Castrof, Oscar Mauricio Gomezg, Sandro Antonio Pereirah, Edvar Yuri Pacheco 7 Schubachi, Kamila Peres Gomese, Heidi Mavengerej, Lucas Gomes de Brito Alvese, Joaquim 8 Lucasa, Hugo Costa Paese, Patricia Albuquerquel, Laurício Monteiro Cruzm, Juan G. McEweng, 9 Jason E. Stajichn, Rodrigo Almeida-Paesh, Rosely Maria Zancopé-Oliveirah, Daniel R. Matutej, 10 Bridget Barkero, Maria Sueli Soares Felipea,p, Marcus de Melo Teixeiraa,e,*, André Moraes 11 Nicola a,e,* 12 13 a Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasília, 14 Brasília, Brazil.

15 b Oswaldo Cruz Institute – Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Brazil.

16 c University Hospital of Brasília, Brasília, Distrito Federal

17 d Laboratório Especial de Ciclo Celular, Butantan Institute, São Paulo, Brazil 18 e Faculty of Medicine, University of Brasília, Brazil

19 f Faculty of Agronomy and Veterinary Medicine, University of Brasília, Brasília, Brazil.

20 g Cellular & Molecular Biology Unit, Corporación para Investigaciones Biológicas, Medellín, 21 Colombia 22 h Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation, Rio de 23 Janeiro, Brazil.

24 I Coordination of Public Health Laboratories, Ministry of Health, Brasília, Brazil. 25 j Department of Biology, University of North Carolina, Chapel Hill, US. 26 l Faculty of Ceilândia, University of Brasília, Brasília, Brazil. 27 m Office of Environmental Surveillance of Zoonoses, Federal District Health Secretariat, 28 Brasília, Brazil.

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

29 n Department of Microbiology & Plant Pathology and Institute for Integrative Genome Biology, 30 University of California, Riverside, Riverside, USA. 31 o Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, USA 32 p Institute of Biological Sciences, University of Brasília, Brasília, Brazil.

33 * These authors share senior authorship 34 35 Corresponding authors: 36 Name: André Moraes Nicola 37 Address: Campus Universitário Darcy Ribeiro, Núcleo de Medicina Tropical, sala 40. Brasília- 38 DF, Brazil. 70910-900. 39 Phone: +55 61 992619048 40 E-mail: [email protected] 41 42 Name: Marcus de Melo Teixeira 43 Address: Campus Universitário Darcy Ribeiro, Núcleo de Medicina Tropical, sala 40. Brasília- 44 DF, Brazil. 70910-900. 45 Phone: +55 61 993992827 46 E-mail: [email protected] 47 48 A novel Sporothrix brasiliensis genomic variant in Midwestern Brazil: 49 evidence for an older and wider sporotrichosis outbreak 50

51 Sporotrichosis is a subcutaneous infection caused by fungi from the genus Sporothrix. The

52 disease is transmitted by inoculation of infective particles found in plant-contaminated material

53 or diseased animals, characterizing the classic sapronotic and emerging zoonotic transmission,

54 respectively. Since 1998, Brazil has experienced a zoonotic sporotrichosis epidemic due to S.

55 brasiliensis, centered in the state of Rio de Janeiro. Our observation of feline sporotrichosis cases

56 in Brasília (Midwestern Brazil), around 900 km away from Rio de Janeiro, led us to question medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

57 whether the epidemic caused by S. brasiliensis has spread from the epicenter in Rio de Janeiro,

58 emerged independently in the two locations, or whether the disease has been present and

59 unrecognized in Midwestern Brazil. A retrospective analysis of 91 human and 4 animal cases

60 from Brasília, ranging from 1993 to 2018, suggests the occurrence of both sapronotic and

61 zoonotic transmission. Molecular typing identified S. schenckii as the agent in two animals and

62 all seven human patients from which we were able to recover clinical isolates. However, in two

63 animals, the disease was caused by S. brasiliensis. Whole-genome sequence typing of seven S.

64 brasiliensis strains suggests that isolates from Brasília are genetically distinct from those

65 obtained at the epicenter of the outbreak in Rio de Janeiro, both in phylogenomic and population

66 genomic analyses. The two S. brasiliensis populations seem to have separated 2.24 - 3.09 million

67 years ago, indicating independent outbreaks or that the zoonotic S. brasiliensis outbreak might

68 have started earlier and be spread wider in South America than previously recognized.

69 70 Keywords: Sporothrix, Sporotrichosis, Sporothrix brasiliensis, Sporothrix schenckii, 71 Zoonotic transmission, Brasília. 72

73 Introduction 74 The emergence of fungal pathogens worldwide is an important and frequently neglected 75 public health issue [1]. One of the most frequent genera of pathogenic fungi is Sporothrix 76 (Sordariomycetes, Ascomycota), which causes the subcutaneous granulomatous disease called 77 sporotrichosis. [2,3]. The etiologic agent of the disease, S. schenckii sensu lato, was recently 78 described to harbor three cryptic species: S. schenckii sensu stricto, S. globosa, and the emerging 79 species S. brasiliensis [4-6]. The three species are saprophytes and produce sympodial and 80 sessile conidia that switch their morphology to the pathogenic yeast phase after a transcutaneous 81 injury [7]. The three species differ in their virulence. S. brasiliensis is more virulent in animal 82 models than S. schenckii and S. globosa, and causes localized to disseminated sporotrichosis that 83 can be fatal to both humans and cats [8]. Lower antifungal susceptibility and refractory medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

84 sporotrichosis occurs in some Brazilian cases related to the zoonotic epidemics [9,10]. Previous 85 population genetics surveys revealed that S. brasiliensis is largely clonal and the little variation 86 in the species is associated to particular geographic areas [6]. On the other hand, its sister taxon 87 S. schenckii shows higher rates of recombination and little to no population structure among 88 Brazilian strains [6]. 89 Currently, sporotrichosis occurs endemically globally but it is especially prevalent in 90 Latin America, South Africa, Madagascar, China, India, and Japan [3]. In addition to endemic 91 disease, multiple sporotrichosis outbreaks have been reported in the last century [2,11,12]. 92 Before the 1990s, sporotrichosis was considered a disease of low-to-moderate endemicity in 93 Brazil. In the past two decades, though, it has become an urgent health problem [7,13]. In 1997, 94 three individuals from the same family got infected by Sporothrix sp. after successive feline 95 zoonotic transmission events in Rio de Janeiro, Brazil [14]. Since 1998, over 5,100 cats have 96 been diagnosed with sporotrichosis in the state of Rio de Janeiro; >5,000 human cases were 97 reported in 2015, suggesting high prevalence of the disease in the state [15]. Rio de Janeiro is 98 currently considered one of the three (along with China and South Africa) sporotrichosis 99 hyperendemic regions [14,16,17], with a crucial difference in incidence of feline sporotrichosis 100 and zoonotic transmission from cats to humans [6,18]. 101 Infections with S. brasiliensis have recently been described in Argentina and Paraguay, 102 the first reports of this species outside Brazil [19,20]. Sporotrichosis caused by S. brasiliensis has 103 recently been described in Northeast Brazil, also mostly due to zoonotic transmission [21-24]. 104 The recent emergence of S. brasiliensis-associated sporotrichosis in places with few or no cases 105 reported in the early 2000s raises the question of whether the outbreak has recently spread from 106 Rio de Janeiro or zoonotic sporotrichosis cases had been happening there, unrecognized. This 107 question is very important in determining public health policy do deal with emergent 108 sporotrichosis. In this work, prompted by our observation in 2015 of a case of sporotrichosis in a 109 cat whose owner also had a lesion that was suggestive of the disease, we address this question. 110 To look for evidence of human zoonotic infections, we documented additional animal 111 cases and retrospectively studied a series of sporotrichosis cases in the University Hospital of 112 Brasília. We also sequenced the genomes of seven Sporothrix spp. strains and compared them to 113 the reference S. brasiliensis 5110 genome [25]. We report for the first time the occurrence of S. 114 brasiliensis infections in animals at the capital of Brazil, Brasília, located in the Midwestern medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

115 portion of the country. We also found a surprising genomic diversity in the S. brasiliensis strains 116 in Brasília, which suggests that independent outbreaks caused by S. brasiliensis occur and might 117 have started earlier and be spread wider than previously recognized. 118

119 Material and Methods 120 Human cases 121 We carried out a cross-sectional study of a series of sporotrichosis cases in the University 122 Hospital of Brasília, from 1993 to 2018. This study was previously approved by the Ethics 123 Committee of the Faculty of Medicine, University of Brasília (protocol CAAE: 124 873718.0.00005558). The inclusion criterion was diagnosis of sporotrichosis by culture of 125 Sporothrix sp. in the Mycology Laboratory of the University Hospital of Brasília. Diagnoses 126 were performed by growing the fungus from biological samples (skin lesions, sputum, broncho- 127 alveolar lavage, and cerebrospinal fluid) on solid culture media (i.e. Sabouraud and Mycosel) 128 followed by microscopic characterization of colonies. The following data were anonymously 129 retrieved from patient medical records: clinical form of sporotrichosis (cutaneous, mucosal and 130 extracutaneous forms), address, year of diagnosis, age, sex, gender, race, occupation, treatment, 131 and disease outcome. 132 133 Animal cases 134 Three cats and one dog with suspected sporotrichosis from the Veterinary Hospital of the 135 University of Brasília, the Directorate for Environmental Surveillance (zoonosis division) of the 136 Federal District Health Department and a private veterinary clinic were referred to the Mycology 137 Laboratory of the University Hospital of Brasília for diagnosis between 2015 and 2018. We 138 performed direct microscopic observation of patient samples and cultured skin lesion specimens 139 as above, followed by microscopic identification of the fungus. Relevant clinical and 140 epidemiological data were also collected to describe the veterinary cases. Animal observations 141 and tests were previously approved by the University of Brasília Ethics Committee on the Use of 142 Animals (CEUA-UnB), protocol: 66716/2016. 143 medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

144 Morphological studies 145 Seven isolates from humans and four isolates from animal cases were used for 146 morphological and molecular characterization. Pure cultures were kept on Sabouraud dextrose 147 agar at room temperature for the development and identification of the mycelial phase. The slide 148 culture method was used to characterize microscopic filamentous features of the Sporothrix spp. 149 isolates. Mycelia fragments were inoculated into 1 x 1 cm Sabouraud dextrose agar and malt 150 extract agar blocks, which were then mounted on a slide, covered with a coverslip and incubated 151 for 14-21 days at room temperature. To induce the yeast phase, the mycelial fragments were 152 inoculated into Brain-Heart Infusion broth and incubated for 7 days at 37 °C and 150 rpm 153 agitation. Both mycelial and yeast preparations were stained with lactophenol blue and 154 visualized with a Zeiss AxioObserver Z1 microscope equipped with a 40X objective. Images 155 were collected using the Zeiss ZEN software. 156 157 Molecular typing and identification 158 We extracted genomic DNA using the PureLink Genomic DNA Mini Kit 159 (ThermoFisher). We started with 500 mg of mycelial tissue as input material. We used glass 160 beads protocol with a Precellys 24 instrument (Bertin). We assess the DNA integrity by running 161 5 μL aliquot of the extraction on a in a 0.8% agar agarose-gel electrophoresis stained with 0.5 162 µg/ml ethidium bromide. To estimate the DNA yield were we used a NanoDrop 1000c 163 Instrument (ThermoFisher). We then amplified a segment of the calmodulin gene with PCR and 164 Sanger sequenced in an ABI 3130xl Instrument (Applied Biosystems). PCR setting and cycling 165 conditions were as previously described [6]. Initial nucleotide BLAST analysis (BLASTn) was 166 performed for each amplicon in order to retrieve the best hit results for each strain on the NCBI 167 database. All extractions (N=15) showed Sporothrix spp. as the closest BLAST match. 168 169 Whole genome sequencing 170 We selected six S. brasiliensis (A001, A005, s15677, s28606, s34180 and s48605) and 171 one S. schenckii (A003) isolates for Illumina short read whole-genome sequencing. The two S. 172 brasiliensis strains, A001 and A005, were retrieved from the collection at the Mycology 173 Laboratory of the University Hospital of Brasília and labelled as SbFD (S. brasiliensis, Federal 174 District). Isolates s15677, s28606, s34180 and s48605 were from the Collection of Pathogenic medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

175 Fungi at the Oswaldo Cruz Foundation, were obtained in the state of Rio de Janeiro and were 176 designated as SbRJ. From each isolate, we extracted 1 µg of DNA, as described above. Next, we 177 prepared paired-end libraries using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina. 178 The libraries were quantified using the NEBNext® Library Quant Kit for Illumina® (New 179 England Biolabs). We pooled equimolar amounts of each library together and sequenced them 180 using in an Illumina NextSeq 550 Instrument using the NextSeq 500/550 High Output Kit v2 181 chemistry. The run was performed in a high output, 2 X 150 bp mode. We used Trimmommatic 182 [26] to remove adapters and filter low-quality bases. 183 184 Public data 185 We used three additional sets of Illumina paired-end reads from S. schenckii isolates from 186 Colombia (EM7 and MS1 - [27]) and Puerto Rico (ATCC 58251 - [28]). To root phylogenetic 187 trees (see below), we retrieved additional genomic assemblies from other Sporothrix species 188 from the NCBI genome browser (Supplementary Table 1). 189 190 Read mapping and variant calling 191 We mapped the newly-sequenced strains and the publicly available data to the S. 192 brasiliensis 5110 reference genome [29] using bwamem. To identify Mismatch intervals and 193 indels, we used GATK v3.3-0RealignerTargetCreator and IndelRealigner [30,31]. To identify 194 polymorphic sites (single nucleotide polymorphisms, SNP), we used the GATK 195 UnifiedGenotyper module using the parameter het = 0.01 to account for a haploid organism. We 196 used the following filters to obtain SNPs calls: QD = 2.0 || FS_filter = 60.0 || MQ_filter = 30.0 || 197 MQ_Rank_Sum_filter = -12.5 || Read_Pos_Rank_Sum_filter = -8 [32]. We excluded 198 polymorphic sites with less than 10X coverage, with less than 90% variant allele calls, or that 199 were identified by Nucmer [33] as located in duplicated regions in the S. brasiliensis reference 200 genome. We identified 115,488 SNPs across five Sporothrix species (16 isolates). 201 202 Phylogenetic tree and whole-genome alignments 203 We used the WGS dataset described above to build a phylogenetic tree of the 16 204 Sporothrix spp. isolates. We used IQTREE [34] to build a Maximum Likelihood (ML) tree with 205 the –m TEST function to determine the best nucleotide substitution model. To assess the strength medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

206 of the support for each clade present in the tree, we found the consensus phylogenomic tree of 207 1,000 ultrafast bootstraps coupled with 1,000 Shimodaira–Hasegawa-like approximate likelihood 208 ratio tests (SH-aLRT) [35,36]. We visualized the consensus topology and branch support with 209 FigTree v1.4.2 – http://tree.bio.ed.ac.uk/software/figtree/. We used the Automatic Assembly For 210 The Fungi (AAFTF) pipeline to assemble the newly-sequenced S. brasiliensis genomes, with 211 default parameters (https://github.com/stajichlab/AAFTF). The S. brasiliensis assemblies with 212 the highest N50 (Supplementary Table 1) were aligned to the strain 5110 reference genome using 213 the DGENIES online tool via minimap2 algorithm for comparative genomic purposes. 214 215 Timing analysis 216 Next, we estimated the approximate divergence time of the splits observed in the 217 phylogenomic analysis using Bayesian inference in BEAST v1.10.4 [37]. To avoid 218 overestimation of branch lengths which would tend to inflate molecular rates, a number of 219 invariable sites were input in the .xml files according to their frequency in the species genome’s 220 composition and their base count of each type (A, C, G, and T) in the respective genome. We 221 used a GTR model with gamma variation among sites (using four discrete classes). Priors for the 222 different dating parameters included: a Normal distribution in which a conservative 90% 223 confidence interval was within 3.8 - 4.9 MYA (following [25]) marking the separation between 224 S. brasiliensis and S. schecnkii; a rate interval of [0.9E-3, 16.7E-3] substitutions/site/branch/MY 225 for the parameter ucld.mean (mean of the uncorrelated lognormal distribution of rates across 226 branches, instead of assuming a more simplistic global clock prior), based on evolutionary rate 227 estimation based on different groups of fungi [38]; and a birth-death tree prior with incomplete 228 sampling [39]. We used two independent Markov Chain Monte Carlo (MCMC) chains each run 229 until convergence; the marginal distributions and traces from the two replicate chains were 230 inspected in Tracer v1.7 [40]. We summarized the two MCMC runs using LogCombiner 231 (BEAST package) after discarding the burnin region. Finally, we used Treeannotator (BEAST 232 package) to annotate clade posteriors, divergence times, and rate variation across branches. 233 234 Population genetic analysis 235 We estimated the average nucleotide diversity (π) within species or populations and absolute

236 differentiation (Dxy) between S. brasiliensis and S. schenckii, and between the S. brasiliensis medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

237 SbFD and SbRJ using a series of Python scripts available at 238 https://github.com/simonhmartin/genomics_general.

239 Results 240 241 Both sapronotic and zoonotic transmissions of sporotrichosis occur in Brasília and 242 surrounding areas 243 The study population involved 91 human cases ranging from 1 to 82 years of age with 244 sporotrichosis confirmed by isolation of Sporothrix spp. in culture at the Mycology Laboratory 245 of the University Hospital of Brasília. All patients were diagnosed either by direct exam or by 246 the microbiological isolation of the pathogen from 1993 to 2018. We reported 64 cases (70%) in 247 males and 27 (30%) females. The years with most cases reported were 1999, 2000, and 2009 248 with 12 (13%), 7 (8%), and 8 (9%) cases, respectively. Regarding age groups, the 21-30- and 51- 249 60-year ranges have more cases in this retrospective study, corresponding to 17 (18.7%) and 16 250 (17.6%) cases, respectively (Table 1). Most patients were treated with itraconazole alone or in 251 association with saturated potassium iodide and, in severe cases, fluconazole and/or 252 amphotericin B were used. 253 We were able to analyze 48 full medical records of the included patients. Among them, 254 34 were presumably infected in Brasília (70.9%) and six in the neighboring state of Goiás 255 (12.5%). Most patients had the lymphocutaneous form (n=34 – 70.8%), followed by fixed 256 cutaneous form (n=6 – 12.6%). Five extra-cutaneous cases were detected in the central nervous 257 system (n=1 – 2%) and or the upper airway tract (n=4 - 8.3%). The clinical presentation of three 258 patients (6.3%) was inconclusive or not reported. The most frequent profession/occupation was 259 student (n=26 - 29%), followed by farmers and retirees with 15% (n=14) and 14% (n=13), 260 respectively (Table 1). Some risk factors we found were prostate and breast cancer, high blood 261 pressure, diabetes, cirrhosis, tuberculosis, leishmaniosis, hepatitis and other chronic diseases. 262 Chagas disease and paracoccidioidomycosis co-infections were found in two sporotrichosis 263 patients. Three cases of HIV coinfection were also diagnosed. Three cases were fatal, two of 264 which in patients with cancer and one with chronic cirrhosis. 265 Additionally, a thorough investigation of the clinical records suggested two main forms 266 of Sporothrix infections in Brasília: (1) the classical sapronotic form acquired from a direct medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

267 inoculation of plant-derived material harboring the fungus, which was more observed in rural 268 and construction workers, and (2) the zoonotic transmission triggered by skin injuries provoked 269 by infected cat or dog bite/scratches. Sapronotic sporotrichosis was reported in 5 patients (10%) 270 after injuries with plant-derived material. Five patients (10%), including two veterinarians, 271 reported cat/dog bites or scratches and six other patients (12.6%) reported living and having 272 frequent contact with dogs or cats with skin lesions. Other patients either did not report any 273 previous injuries or reported a different exposure source (i.e. sporotrichosis acquired after 274 injuries with barbed wire and metal cans). 275 In parallel, we diagnosed, from 2015 to 2018, 4 animals (3 cats and 1 dog) with 276 sporotrichosis in Brasília. These animals had classical clinical presentations such as multiple 277 suppurative granulomatous cutaneous lesions with a high load of yeast cells, generally with 278 involvement of mucous membranes. Lesions were commonly found at parts such as the head, 279 especially on the nose, as well as on the chest and legs. Two felines were euthanized and the 280 other cat and the dog were treated with itraconazole and potassium iodide. Lastly, the pet owner 281 of the cat in which the A001 strain was recovered, was also diagnosed with cutaneous 282 sporotrichosis. The diagnosis and treatment were made in another hospital in Brasília, so we do 283 not have medical records or a clinical isolate obtained from the owner. 284 285 Sporothrix brasiliensis isolates from Brasília and the epicenter of the zoonotic sporotrichosis 286 epidemics, Rio de Janeiro, are genetically distinct. 287 In order to differentiate between two different hypotheses for S. brasiliensis presence in 288 Brasília, that it was recently introduced here from Southeast Brazil or that the Brasília isolates 289 are actually endemic to that region, we performed a number of genetics and genomics analyses. 290 First, we inferred the genealogical relationships between isolates of different species of 291 Sporothrix using whole genome sequencing. By rooting our tree with S. insectorum, we observed 292 that S. schenckii and S. brasiliensis are sister species and form a triad along with S. globosa 293 (Figure 2). The Most Recent Common Ancestor (tMRCA) of Sporothrix emerged between 26.63 294 and 37.38 Million Years Ago (MYA) (Figure 3). Unlike S. insectorum and S. pallida, this triad 295 of species cause sporotrichosis in humans and animals and form the “Sporothrix pathogenic 296 clade”. The crown age of this pathogenic group (TMRCA) is between 4.24 - 5.28 MYA. The medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

297 divergence between the pathogenic clade and S. pallida occurred 10.22 - 14.09 MYA, suggesting 298 that the pathogenesis syndrome must have arisen in the last 15 MY. 299 Our WGS phylogenetic tree also allows us to assess the partition of the genetic variation 300 within S. schenckii. We found support for the existence of two clades, one harboring two 301 Colombian isolates (SsEM7 and SsMS1) and a cat-derived isolate from Brasília (A003) and a 302 clade composed by one strain from the USA (1099-18) and an additional strain from Puerto Rico 303 (ATCC 58251). We estimate that divergence of these two groups occurred between 4.42 and 304 5.82 MYA. These results suggest that South American and North American populations of S. 305 schenckii are genetically differentiated and call for the question of whether there has been 306 speciation associated with geographical distribution within S. schenckii. 307 Next, we studied the relationships between the S. brasiliensis isolates from Brasília and 308 Rio de Janeiro. The five Rio de Janeiro isolates (5110, s15677, s28606, s34180 and s48605) 309 form a monophyletic group that does not include the two Brasília isolates, A001 and A005, 310 which in turn are clustered in their own group. We also compared genomic similarities between 311 the two S. brasiliensis cluster variants by aligning one individual from each cluster to the 5110 312 reference strains using the minimap2 tool. By aligning the S. brasiliensis strain s15677 to the S. 313 brasiliensis 5110 reference strain (both belonging to the Rio de Janeiro population), we observed 314 that the genomes are 99.81% similar. By aligning the S. brasiliensis A001 strain to the same 315 reference, the genomic similarity dropped to 96.31% suggesting that those two groups are 316 genetically unrelated (Supplementary Figure 1). 317 We calculated the approximate divergence time between S. brasiliensis isolates from 318 Brasília and Southeastern Brazil. The TMRCA of the S. brasiliensis species was estimated to be 319 2.24 - 3.09 MYA, the TMRCA for the strains from Rio de Janeiro is 1.94 - 2.7 MYA, and the 320 TMRCA for the strains from Brasília was 0.52-0.93 MYA (Figure 3). The S. brasiliensis isolates 321 A001 and A005 are clustered in a single branch and are phylogenetically different from those 322 isolated in Rio de Janeiro (5110, s15677, s28606, s34180 and s48605 – Figure 2). This result 323 suggests that those infections were likely to be locally acquired in Brasília, not in Rio de Janeiro. 324 These results suggest that the two populations are genetically distinct and that the infections 325 observed in Brasília are not the result of a small group of Rio de Janeiro isolates that migrated. 326 Since the infections caused by S. brasiliesis in Brasília happened autochthonously, the causal medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

327 agent of these infections is genomically differentiated from the causal agent of the Rio de Janeiro 328 infections. 329 We evaluate a second prediction of the hypothesis that Rio de Janeiro isolates caused the 330 Brasília outbreak. The source population of the outbreak should have a larger genetic diversity 331 than the outbreak population because the former has not undergone a bottleneck. We measured 332 the magnitude of genetic variation in each Sporothrix group using pairwise calculations of 333 heterozygosity. S. schenckii has the highest nucleotide diversity (π=1.67%), which is in line with 334 the measurements of other fungal species with complex population composition and worldwide 335 distribution [41,42]. The genetic variation within S. brasiliensis has much lower nucleotide 336 diversity (π=0.52%) than that of its sister species, probably due to its endemicity to South 337 America. We next calculated the nucleotide diversity within the two Brazilian S. brasiliensis 338 groups. The Brasília group has a nucleotide diversity on the same order of magnitude as S. 339 brasiliensis as a whole (π=0.20%). On the other hand, the Rio de Janeiro population has an 340 extremely low genetic diversity (π=9.78 ×10-4%), suggesting an extreme bottleneck. In 341 agreement with our inference from the phylogenomic tree, these results indicate that the Brasília 342 population is extremely unlikely to be derived from the Rio de Janeiro one. 343 Our phylogenetic tree suggested genetic differentiation between S. brasiliensis isolates

344 from Rio de Janeiro and Brasília. We calculated the pairwise absolute genetic distance (Dxy)

345 between S. schenckii and S. brasiliensis, and within S. brasiliensis. The Dxy between those two 346 species was 0.042 while between the Brasília and Rio de Janeiro populations it was 0.010. Figure 347 3 shows the mean pairwise differences per site within populations (nucleotide diversity or π) and

348 mean pairwise differences per site between populations Dxy along the 13 scaffolds of the 349 Sporothrix genome. The genome differentiation between S. schenckii and S. brasiliensis is 350 extensive and widespread along the whole genome. We observed a strikingly different pattern 351 when we compare the two populations of S. brasiliensis. As expected, Rio de Janeiro S.

352 brasiliensis has little genetic variation along the whole genome (Figure 3A). The Dxy along most 353 of the genome is low suggesting low differentiation between Rio de Janeiro and Brasília despite 354 almost 1 MY of divergence (See above, Figure 2). Nonetheless, scaffolds I, III, V, IX and XI,

355 show peaks of high differentiation. Notably, the Dxy peaks in scaffolds I, III, and XI are highly 356 differentiated between Rio de Janeiro and Brasília S. brasiliensis and between S. brasiliensis (as 357 a whole) and S. schenckii. These results suggest that the differentiation between Brazilian medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

358 populations of S. brasiliensis is modest and localized and not as pronounced as between S. 359 schenckii and S. brasiliensis. This difference might simply stem from the difference in 360 divergence time between the Sporothrix species and the S. brasiliensis populations.

361 Discussion 362 Sporotrichosis due to S. brasiliensis has become one of the most important fungal 363 diseases in Brazil. The disease has gone from a localized outbreak in Rio de Janeiro state to a 364 country-wide epidemic during the past 20 years and has affected thousands of humans and 365 animals. In Brasília, located in Midwestern Brazil and roughly 900 km from Rio de Janeiro, we 366 observed indications that suggest cat/dog-transmitted sporotrichosis might occur. Moreover, both 367 cats and dogs are affected by pathogenic Sporothrix spp. In Rio de Janeiro, both fungal species 368 have been isolated from dogs [18], but S. brasiliensis has been identified in almost all cats tested 369 so far [16]. The etiologic agent of this disease was recently found in clinical and environmental 370 samples in Argentina as well in veterinary samples in Paraguay [19,20]. 371 In this study, we dissect the support for two different hypotheses for the expansion of this 372 disease throughout South America, namely that S. brasiliensis recently (< 20 years) spread from 373 Rio de Janeiro to other areas in Brazil and other countries via human, animal hosts or fomites; or 374 that different groups of S. brasiliensis occur in different locations of the South American 375 continent. We focused on a recent sporotrichosis case series in Brasília. If the former hypothesis 376 is correct, one would expect that the Rio de Janeiro population would have a larger genetic 377 variation than the Brasília population. Additionally, one would expect the Rio de Janeiro 378 population to encompass the genetic variants observed in Brasília. Neither of these were 379 observed in our data. The nucleotide diversity (π) in Rio de Janeiro is 200 times lower than in 380 Brasília. Additionally, the Rio de Janeiro and Brasília populations are reciprocally monophyletic 381 which contradicts the idea that Brasília strains are a subset of the Rio de Janeiro group. 382 Moreover, the split time between Brasília and Rio de Janeiro occurred between 0.52-0.93 MYA, 383 predating the human colonization of the Americas and thus precluding the possibility that S. 384 brasiliensis migrated associated with humans. 385 The observation that the surge on sporotrichosis in Brasília is not caused by recent 386 migration from Rio de Janeiro poses two additional questions. First, other reasons, besides 387 migration must be responsible for the recent uptick of cases. One possibility is that deforestation 388 and human expansion into previously uninhabited areas has put humans (and their pets) in medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

389 contact with fungi that otherwise had never infected humans. A second possibility is that in the 390 last 20 years, a previously tamed lineage of S. brasiliensis became more virulent. Both novel 391 mutation and introgression from a virulent lineage could be responsible. Finally, it is possible 392 that the improvement of epidemiology and medical observations has led to the identification and 393 recording of sporotrichosis due to S. brasiliensis. 394 Second, it remains unclear why the number of cases in Brasilia is lower than that in Rio 395 de Janeiro, one of the most hyperendemic areas in the world. One potential explanation for the 396 higher incidence of sporotrichosis in Rio de Janeiro than Brasília is higher population density 397 and less favorable sanitation conditions in the former area [17]. Our study shows that cats can 398 acquire sporotrichosis caused by both S. schenckii and S. brasiliensis, which indicates that feral 399 and stray cats can be reservoirs of the disease. Another possibility is that Brasilia and Rio de 400 Janeiro differ in their number of outdoor cats. Studies have shown a high prevalence of 401 parasitosis in cats from both locations (Brasilia: [43], RJ:[44]) but the number of felines and their 402 relative health conditions remain unknown. 403 Our work suggests the existence of at least two groups of S. brasiliensis in South 404 America. The increase in sporotrichosis incidence, along with the increments on the disease 405 burden of other mycoses across the world, and the recent outbreak of Cryptococcus gattii - 406 originated in the Amazon region - in the Pacific Northwest [45] are sobering indications of the 407 importance of efforts to detect and mitigate or prevent the spread of fungal emerging diseases, 408 both in South America and elsewhere. 409 410 Acknowledgments 411 412 The authors would like to thank to Jéssica S. Boechat and Manoel M. E. Oliveira 413 (Fiocruz) for the technical support. A.M.N was funded by FAP-DF awards 0193.001048/2015- 414 0193.001561/2017 and the CNPq grant 437484/2018-1. B.M.B. was supported by NIH/NIAID 415 award R21AI28536. D.R.M. was supported by NIH/NIGMS award R01GM121750. M.M.T was 416 supported by CNPq/UNIVERSAL award 43460/2018-2. M.S.S.F was supported by FAP- 417 DF/PRONEX award 193.001.533/2016. S.A.P. was supported by FAPERJ, grant number E- 418 26/202.737/2019. 419 medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

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539 Tables 540 Table 1 – Epidemiological characteristics of sporotrichosis in Brasília, Brazil

Gender (n=91) Male 64 (70.32%) Female 27 (29.68%) Profession (n=91) Students 26 (28.57%) Rural Worker 14 (15.38%) Household/retired 13 (14.28%) Veterinary 2 (2.2%) Other 4 (4.39%) Not reported 32 (35.18%) HIV condition (n=91) positive 3 (3.29%) negative 88 (96.71%) Age Group (n=91) 0 - 10 12 (13.19%) 11 - 20 10 (10.99%) 21 - 30 17 (18.68%) 31 - 40 11 (12.09%) 41 - 50 11 (12.09%) 51 - 60 16 (17.58%) >61 14 (15.38%) Sporotrichosis forms (n=48) Cutaneous fixed 6 (12.5%) Lymphocutaneous 34 (70.83%) Disseminated 5 (10.42%) Unknown 3 (6.25%) Source of infection (n=48) medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Living with a cat or dog 6 (12.25%) 541 Cat/dog bite or scratch 5 (10.42%) 542 Trauma with plant/wood material 5 (10.42%) Unknown 32 (66.91%) medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

543 Figures

544 545 Figure 1 – Whole-genome Sporothrix genealogy using BEAST analyses. The phylogenomic tree 546 was inferred using Bayesian inference under the GTR model with gamma variation among sites 547 and S. insectorum was set as the outgroup. The 90% confidence interval marking the separation 548 between S. brasiliensis and S. schenckii was set to 3.8 - 4.9 MYA, a rate interval of [0.9E-3, 549 16.7E-3] based on evolutionary rate estimation based on different groups of fungi and a birth- 550 death tree prior with incomplete sampling were set as priors. The clade distribution, posterior 551 probabilities and divergence times are shown. medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

552 553 Figure 2 – Population genetics plots along the S. brasiliensis 5110 reference genome. We have 554 calculated the mean nucleotide diversity (π) within species or populations and absolute

555 differentiation (Dxy) between S. brasiliensis and S. schenckii, and between the S. brasiliensis 556 SbFD and SbRJ (red line) and plotted along each scaffold of the S. brasiliensis 5110 genome 557 using a 5kb window. medRxiv preprint doi: https://doi.org/10.1101/2020.07.09.20142125; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

558 559 Supplementary Figure 1 – Dot plots of whole genome alignments between different S. 560 brasiliensis genotypes. The S. brasiliensis strains s15677 (A - RJ population) and A001 (B – FD 561 population) were aligned to the S. brasiliensis 5110 reference strain and the similarity values are 562 displayed for each scenario.